We seek funding for a pilot X-ray fine structure experiment on the metal sites in alkaline phosphatase to determine accurate zinc-zinc bond distances in the active site and location of the substrate binding site. This ubiquitous metalloenzyme serves as an important medical diagnostic tool in mammalian tissues, with different isozymes appearing in bone, intestine, kidney membranes, and in blood and milk. The proposed experiments focus on well-characterized Zn and Mg E. Coli prototype alkaline phosphatase for which the recent 4A x-ray map discloses the six metal binding sites as earlier implied from NMR, ESR and optical studies over the last decade. A detailed bonding picture will serve as the basis for understanding such unresolved problems as active site partial occupancy, gross conformational changes on substrate binding, and positive and negative co-operativity between metal and substrate binding sites. Enzymes with known metal content will be prepared and characterized at the P.I.'s laboratory, and X-ray metal edge spectra will be taken at the Cornell Synchrotron Radiation Source (CHESS) in a collaborative project. If successful, these types of experiments can be amplified to other well-characterized chemically modified variants of alkaline phosphatase, and thereby clarify the nature of the acid denaturation and reassembly cycle of this important enzyme, its allosteric control and other structure-function controversies.